CN113884680A - Carbon quantum dot fluorescence immunoassay rapid detection kit and detection method for six veterinary drug antibiotics in poultry tissues - Google Patents
Carbon quantum dot fluorescence immunoassay rapid detection kit and detection method for six veterinary drug antibiotics in poultry tissues Download PDFInfo
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Abstract
The invention discloses a carbon quantum dot fluorescence immunoassay rapid detection kit and a detection method for six veterinary drug antibiotics in poultry tissues, and belongs to the field of fluorescence immunoassay. Comprises a box body, a reaction cup arranged in the box body and a reagent arranged in the box body; the kit for the rapid fluorescence immunoassay comprises a reaction cup and a fluorescence immunoassay kit, wherein the reaction cup comprises 100 reaction cups respectively coated with furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol coating antigens.
Description
Technical Field
The invention relates to a small molecule to-be-detected substance carbon quantum dot fluorescence immunoassay rapid detection kit and a detection method thereof, belonging to the technical field of immunoassay.
Background
Under the promotion of nanotechnology, fluorescent materials such as semiconductor Quantum Dots (QDs), up-conversion nanomaterials (UCNPs) and long-afterglow nanomaterials are applied to the establishment of fluorescence quenching immunoassay methods to meet different analysis requirements. However, the problems of large heavy metal toxicity of QDs, few fluorescence types of UCNPs and the like become critical limiting factors of the QDs and the UCNPs. Carbon quantum dots (CDs), as a class of zero-dimensional (OD) nanomaterials with excellent photoluminescence properties, are fluorescent nanomaterials that have been widely studied since the successful synthesis of semiconductor quantum dots. The elemental composition of CDs, which was first proposed in 2004, is different from conventional quantum dots, which are typically composed of a heavy metal core. CDs are substantially free of metallic elements, and have the advantage of low toxicity. In addition, it generally exhibits Photoluminescence (PL) behavior that is related to size and excitation wavelength, and can reach PL levels close to those reached by conventional semiconductor Quantum Dots (QDs). Meanwhile, CDs have attracted a great deal of attention as a green substitute for QDs in the fields of biosensing and bioimaging because of their excellent water solubility and biocompatibility. Moreover, CDs also exhibit excellent photobleaching resistance, are inexpensive and easy to manufacture. Therefore, CDs are considered as a novel versatile material in the field of bioluminescence. Mintz et al utilize energy transfer between carbon quantum dots (CDs) and gold nanoparticles (AuNPs) to achieve highly sensitive detection of 3.4 μ nM of alpha-fucosidase through C-point fluorescence quenching degree caused by energy transfer; dong et al quenched the CDs fluorescence by the enzymatic product of horseradish peroxidase/alkaline phosphatase based on the fluorescence internal filtering effect, and converted the absorption signal of conventional ELISA into a fluorescence signal to achieve high-sensitivity detection and analysis of 0.02ng/mL amantadine in chicken samples. The above reports all show that CDs have the capability of becoming a high-sensitivity fluorescence donor probe and realizing high-sensitivity rapid detection and analysis.
Based on the method, the carbon quantum dots are prepared in the aqueous solution environment by a simple method, and a rapid and high-sensitivity fluorescence immunoassay rapid detection kit is established. At present, a carbon quantum dot fluorescence immunoassay rapid detection kit based on comprehensive pretreatment of six veterinary drug antibiotics in poultry tissues is not reported.
Disclosure of Invention
In view of the above, the present invention provides a carbon quantum dot fluorescence immunoassay rapid detection kit for detecting an analyte in an animal derived food and a detection method thereof, and in order to achieve the above object, the technical scheme of the present invention is as follows:
a carbon quantum dot fluorescence immunoassay rapid detection kit for comprehensive pretreatment of six veterinary drug antibiotics in poultry tissues comprises a kit body, a reaction cup arranged in the kit body and a reagent arranged in the kit body; wherein the reaction cups comprise 100 reaction cups respectively coated with furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol coating antigens, and the reagent comprises: furaltadone horse radish peroxidase labeled antibody working solution, furacilin horse radish peroxidase labeled antibody working solution, furazolidone horse radish peroxidase labeled antibody working solution, chloramphenicol horse radish peroxidase labeled antibody working solution and florfenicol horse radish peroxidase labeled antibody working solution; furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol series standard solutions; concentrating the acid sample extracting solution I; concentrating the alkaline sample extracting solution II; 2-nitrobenzaldehyde derivatizing agent; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; a carbon quantum dot fluorescence reaction solution A and a carbon quantum dot fluorescence reaction solution B.
Preferably, the carbon quantum dot fluorescence reaction solution a contains hydrogen peroxide and carbon quantum dots, and the carbon quantum dot fluorescence reaction solution B contains 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
The preparation of the carbon quantum dot comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
The reaction cup is a single-hole or 8-linked reaction cup with the volume of 300 mu L or 500 mu L or other volumes, and the furaltadone, nitrofural, nitrofurantoin, furazolidone, chloramphenicol and florfenicol series standard solutions are respectively obtained by diluting pure furaltadone, nitrofural, nitrofurantoin, furazolidone, chloramphenicol and florfenicol.
A method for detecting an object to be detected by a carbon quantum dot fluorescence immunoassay rapid detection kit of six veterinary drug antibiotics in poultry tissues is characterized in that a corresponding standard solution or a sample complex solution subjected to comprehensive pretreatment and a corresponding enzyme-labeled antibody are respectively added into reaction cups of furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol, the reaction cups are incubated at 37 ℃ for 20min and then washed, a fluorescence reaction solution A and a fluorescence reaction solution B are added, the reaction cups are incubated at 37 ℃ for 10min and then fluorescence values are detected, the inhibition rates are calculated by adding the fluorescence values of standard objects to be detected with different concentrations, the inhibition rates are taken as vertical coordinates, the logarithm of the concentration of the standard objects to be detected is taken as a horizontal coordinate to be taken as a standard curve, and the detection limit and the sensitivity, namely the concentration (IC) of the corresponding object to be detected when the inhibition rates are 50 percent50) The fluorescence value can be read from the standard curve, and is measured at 560nm for each reaction cup with a fluorescence detector at 365nm excitation wavelength.
Preferably, the steps of obtaining the sample complex solution after the comprehensive pretreatment are as follows: (1) weighing 2.0g of sample into a 50mL centrifuge tube, adding 5mL of acidic sample extracting solution I and 150 μ L of 2-nitrobenzaldehyde derivatization reagent, and carrying out vortex oscillation for 5 min; (2) placing the mixture in a water bath kettle at the temperature of 75-80 ℃ and incubating the mixture for 15min in the dark, taking out the mixture and cooling the mixture to room temperature, adding 5mL of alkaline extracting solution II and 8mL of ethyl acetate, carrying out vortex oscillation for 5min at 4000rpm for 5min, taking 2mL of centrifuged supernatant into a 5mL centrifuge tube, and drying the supernatant in air at the temperature of 60 ℃ for 10-15 min; (3) adding 2mL of n-hexane and 1mL of phosphate complex solution, and carrying out vortex for 10s at 4000rpm for 3 min; (4) and taking down the layer to be tested.
The method for calculating the inhibition rate of the kit comprises the following steps of (F)x-F0)/(F0-Fb) X 100% where F0For the detection of the fluorescence value of the cuvette in the case of a standard solution without test substance, FxFor detecting the fluorescence value of the cuvette in the case of a standard solution or a sample solution containing an analyte, FbThe fluorescence value of the reaction cup when the standard solution or sample is not added is determined, the inhibition rate is determined by adding the fluorescence values of the standard substance with different concentrations, the inhibition rate is used as the ordinate, the logarithm of the concentration of the standard substance is used as the abscissa, the standard curve is drawn, and the detection limit and the sensitivity, namely the concentration (IC) of the corresponding substance with the inhibition rate of 50 percent are determined50) Can be read from the standard curve; the concentration of each sample can be calculated by measuring the fluorescence value of the sample to calculate the inhibition rate, and the inhibition rate is read from the standard curve.
Preferably, the detection sensitivity of the kit to furaltadone is 0.12 ng/mL; the detection sensitivity of the furacilin is 0.05 ng/mL; the detection sensitivity of the nitrofurantoin is 0.35 ng/mL; the detection sensitivity of furazolidone is 0.25 ng/mL; the detection sensitivity of the chloramphenicol is 0.12 ng/mL; the detection sensitivity of florfenicol is 0.1 ng/mL.
Preferably, the fluorescence value is measured at 560nm of each reaction cup by a fluorescence detector at an excitation wavelength of 365 nm.
Compared with the existing domestic and foreign micromolecular substance immunoadsorption detection technology, the invention has the following outstanding advantages:
1. the invention uses the carbon quantum dot fluorescence signal to replace the colorimetric signal of enzyme-linked immunoassay, and realizes the high-sensitivity fluorescence immunoassay of the object to be detected;
2. the kit of the invention unifies the pretreatment processes of six targets, the solution of the object to be detected obtained by one treatment process can be used for measuring six components without separate treatment, thereby saving the detection time and reducing the cost and the labor power.
3. The quantity of the reaction cups in the kit can be selected according to the detection requirement, six target objects in 100 samples can be simultaneously and quickly detected and analyzed within 1 hour, and the kit is suitable for quickly screening a large number of samples and can be used as an effective screening means for quickly detecting micromolecule to-be-detected objects in food.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the invention without limitation. In the drawings:
FIG. 1 is a fluorescence spectrum of the carbon quantum dot of the present invention.
FIG. 2 is a result chart of furaltadone detection by ELISA and carbon quantum dot fluorescence immunoassay rapid detection kit.
FIG. 3 is a result chart of furacilin detection by ELISA and carbon quantum dot fluorescence immunoassay rapid detection kit.
FIG. 4 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for nitrofurantoin detection.
FIG. 5 is a result chart of furazolidone detection by ELISA and carbon quantum dot fluorescence immunoassay rapid detection kit.
FIG. 6 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for chloramphenicol detection.
FIG. 7 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for detecting florfenicol.
Detailed Description
The invention will be described in more detail hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown, but the examples are not intended to limit the invention.
Example l (preparation example)
Carbon quantum dot preparation
(1) And (3) synthesis of carbon quantum dots: 3g of citric acid and 5g of urea were added to 10mL of ultrapure water to form a clear solution. The solution was then heated in a 800W microwave oven for 5 minutes during which time the solution changed from a colorless liquid to brown and finally to a dark brown clustered solid. After cooling to room temperature, 20mL of water was added to dissolve the product.
(2) And (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence spectrum shown in FIG. 1, and fluorescence is observed at 400-600 nm under excitation of 365nm excitation light.
Example 2 (preparation example)
(1) Coating antigen: furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol envelope antigens were diluted to 0.25, 0.5 and 0.5. mu.g/mL with pH9.6 sodium carbonate buffer, and 300. mu.L of 100-.
(2) Washing the reaction cup: the reaction solution is discarded the next day, then 200-.
(3) And (3) sealing: add 200. mu.L of casein blocking solution to the reaction cup, incubate at 37 ℃ for 1.5h, discard the casein blocking solution, vacuum seal and store at 4 ℃.
Example 3 (application example)
Use technology of carbon quantum dot fluorescence immunoassay rapid detection kit
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard product diluent containing a substance to be detected with a certain concentration into reaction cups coated with corresponding coating antigens of the substance to be detected, adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substance to be detected into each reaction cup, wherein each concentration is parallel, incubating at 37 ℃ for 20min, removing the reaction solution, adding 200 plus 500 mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the steps for 3 times, and finally, beating the plate on filter paper to dry.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
2. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit inhibits substrate oxidation by combining an object to be detected with an enzyme-labeled antibody so as to cause fluorescence recovery, and the fluorescence intensity value at 560nm in a reaction cup to be detected is increased along with the increase of the content of the object to be detected in a sample to be detected under the excitation of 365 nm. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb)×100％，F0To determine the fluorescence value of the cuvette in a standard solution without test substance, FxFor detecting the fluorescence value of the cuvette in the detection of a standard solution containing an analyte, FbThe fluorescence value detected by the cuvette when the standard solution of the analyte is not added is represented by a standard curve with the inhibition ratio as ordinate and the logarithm of the concentration of the analyte as abscissa, and the sensitivity, i.e., the concentration of the analyte corresponding to the inhibition ratio of 50% (IC)50) Can be read from the standard curve. As shown in FIGS. 2-7, the detection sensitivity of the method of the invention to furaltadone is 0.12 ng/mL; the detection sensitivity of the furacilin is 0.18 ng/mL; the detection sensitivity of the nitrofurantoin is 0.35 ng/mL; the detection sensitivity of furazolidone is 0.25 ng/mL; the detection sensitivity of the chloramphenicol is 0.12 ng/mL; the detection sensitivity of the florfenicol is 0.032 ng/mL.
Example 4 (application example)
Use of enzyme-linked immunoassay
1. Detection step
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard product diluent containing a substance to be detected with a certain concentration into reaction cups coated with corresponding coating antigens of the substance to be detected, adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substance to be detected into each reaction cup, wherein each concentration is parallel, incubating at 37 ℃ for 20min, removing the reaction solution, adding 200 plus 500 mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the steps for 3 times, and finally, beating the plate on filter paper to dry.
(2) Color development: to each reaction cup, 50. mu.L each of a urea peroxide solution and a 3,3',5,5' -tetramethylbenzidine solution was added, and color development was performed at 37 ℃ for 10 min.
(3) And (4) terminating: the color reaction was stopped with 50. mu.L of sulfuric acid stop solution and read within 10 min.
(4) Reading: the absorbance value at 450nm of each reaction cup was measured with a microplate reader.
2. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit disclosed by the invention inhibits substrate oxidation by combining an object to be detected with an enzyme-labeled antibody so as to reduce the absorbance value, and the absorbance value of a solution in a reaction cup to be detected at 450nm is reduced along with the increase of the content of the object to be detected in a sample to be detected. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) ═ a0-AX)/(A0-Ab)×100％，A0To determine the absorbance of the reaction cuvette in the case of a standard solution without test substance, AxFor detecting the absorbance value of a cuvette in a standard solution containing an analyte, AbThe absorbance value of the reaction cup is detected when standard solution of the substance to be detected is not added, the inhibition ratio is used as the ordinate, the logarithm of the concentration of the substance to be detected is used as the abscissa, the standard curve is made, and the sensitivity is the corresponding concentration (IC) of the substance to be detected when the inhibition ratio is 50 percent50) Can be read from the standard curve. As shown in FIG. 2, the sensitivity of the ELISA method of the present invention to furaltadone detection is 0.32 ng/mL; the detection sensitivity of the furacilin is 0.26 ng/mL; the detection sensitivity of the nitrofurantoin is 0.68 ng/mL; the detection sensitivity of furazolidone is 0.36 ng/mL; the detection sensitivity of the chloramphenicol is 0.28 ng/mL; the detection sensitivity of florfenicol is 0.075 ng/mL.
Example 5 (application example)
Use technology of carbon quantum dot fluorescence immunoassay rapid detection kit
1. Pretreatment of avian tissue products
(1) Weighing 2.0g of sample into a 50mL centrifuge tube, adding 5mL of acidic sample extracting solution I and 150 μ L of 2-nitrobenzaldehyde derivatization reagent, and carrying out vortex oscillation for 5 min; a
(2) Placing the mixture in a water bath kettle at the temperature of 75-80 ℃ and incubating for 15min in a dark place, taking out and cooling to room temperature, adding 5mL of alkaline sample extracting solution II and 8mL of ethyl acetate, carrying out vortex oscillation for 5min at 4000rpm for 5min, taking 3mL of centrifuged supernatant into a 5mL centrifuge tube, and drying the supernatant in air at the temperature of 60 ℃;
(3) adding 2mL of n-hexane and 1.5mL of sample complex solution, vortexing for 10s, and centrifuging at 4000rpm for 3 min;
(4) and taking down the layer to be tested.
3. Detection step
(1) Adding a sample to be detected and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of sample extracting solution into reaction cups coated with six substances to be detected, adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substances to be detected into each reaction cup, wherein the concentrations of the two substances are parallel, incubating for 20min at 37 ℃, adding 200-500 mu L of phosphate washing solution after discarding the reaction solution, shaking for 2min, throwing off the phosphate washing solution, repeating the steps for 3 times, and finally drying the plate on filter paper.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
4. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit provided by the invention inhibits the generation of the bottom of a to-be-detected object so as to cause fluorescence reversion, and the fluorescence intensity value at 560nm in a to-be-detected reaction cup is increased along with the increase of the content of the to-be-detected object in a to-be-detected sample under the excitation of 365 nm. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb) X 100% where F0For measuring the fluorescence value of the cuvette for the negative sample of the test object, FxFor detecting fluorescence values of cuvettes for positive samples of test substances, FbThe fluorescence value of the reaction cup without the sample well to be detected is detected. And calculating the inhibition rate by using the fluorescence values of the standard substances to be detected with different concentrations, taking the inhibition rate as a vertical coordinate and the logarithm of the concentration of the standard substances to be detected as a horizontal coordinate to form a standard curve, wherein the concentration of each sample can be calculated by measuring the fluorescence value of the sample to calculate the inhibition rate, and the inhibition rate is read from the standard curve according to the inhibition rate. The kit is used for detecting the furaltadone detection sensitivity of poultry tissue samples0.64 ng/mL; the detection sensitivity of the furacilin is 0.52 ng/g; the detection sensitivity of the nitrofurantoin is 1.36 ng/g; the detection sensitivity of furazolidone is 0.72 ng/g; the detection sensitivity of the chloramphenicol is 0.56 ng/g; the detection sensitivity of the florfenicol is 0.15 ng/g.
TABLE 1 recovery rates of furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol in poultry samples detected by carbon quantum dot fluorescence immunoassay rapid detection kit
As shown in Table 1, the detection value and the addition value of the test substance in the aquatic product sample detected by the kit are consistent.
Claims (10)
1. A carbon quantum dot fluorescence immunoassay rapid detection kit for six veterinary drug antibiotics in poultry tissues is characterized in that the kit comprises reaction cups respectively coated with furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol or florfenicol antigens, furaltadone horse radish peroxidase labeled antibody working solution, furacilin horse radish peroxidase labeled antibody working solution, chloramphenicol horse radish peroxidase labeled antibody working solution and florfenicol horse radish peroxidase labeled antibody working solution, furaltadone, furacilin, chloramphenicol and florfenicol series standard solutions; concentrating the acid sample extracting solution I; concentrating the alkaline sample extracting solution II; 2-nitrobenzaldehyde derivatizing agent; concentrating the phosphate washing solution; concentrating the phosphate compound solution; the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B.
2. The kit for the rapid detection of the carbon quantum dot fluorescence immunoassay of the six veterinary drug antibiotics in the poultry tissues according to claim 1, wherein the reaction cup is a single-hole reaction cup or an 8-linked reaction cup made of polystyrene, and the volume of the reaction cup is 300 μ L or 500 μ L or other volumes.
3. The kit for the rapid fluorescence immunoassay of carbon quantum dots of six veterinary antibiotics in avian tissues according to claim 1, wherein the carbon quantum dot fluorescence reaction solution A comprises hydrogen peroxide and carbon quantum dots, and the carbon quantum dot fluorescence reaction solution B comprises 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
4. The kit for the rapid fluorescence immunoassay of the carbon quantum dots of the six veterinary drug antibiotics in the poultry tissues according to claim 1, wherein the preparation of the carbon quantum dots comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
5. The kit as claimed in claim 1, wherein the kit is characterized in that the kit comprises reaction cups coated with antigens, and the preparation steps comprise diluting furaltadone, nitrofurazone, nitrofurantoin, furazolidone, chloramphenicol and florfenicol coated antigens to 0.25, 0.5 and 0.5 μ g/mL with a pH9.6 sodium carbonate buffer solution, respectively adding 300 μ L of 100 plus materials into each reaction cup, placing in a refrigerator at 4 ℃ for coating overnight, adding 250 μ L of phosphate washing solution into the reaction cups coated with the antigens the next day, shaking for 2min, discarding the phosphate washing solution, repeating the steps for 3 times, finally drying the reaction cups on a filter paper, adding 250 μ L of 100 plus materials into each reaction cup, sealing the reaction cups at 37 ℃ for 1-2h, discarding the casein sealing solution, making into furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol reaction cup, vacuum sealing, and storing at 4 deg.C.
6. The method for detecting the analyte by using the carbon quantum dot fluorescence immunoassay rapid detection kit for six veterinary drug antibiotics in poultry tissues as claimed in claim 1 is characterized in that a corresponding standard solution or a sample complex solution after comprehensive pretreatment and a corresponding enzyme-labeled antibody are respectively added into furaltadone, furacilin, nitrofurantoin, furazolidone, chloramphenicol and florfenicol reaction cups, incubated for 20min at 37 ℃, washed, added with a fluorescence reaction solution A and a fluorescence reaction solution B, incubated for 10min at 37 ℃, and then the fluorescence value is detected, the inhibition rate is calculated by adding the fluorescence value of the analyte standard with different concentrations, the inhibition rate is used as a vertical coordinate, the logarithm of the concentration of the analyte standard is used as a horizontal coordinate to construct a standard curve, and the detection limit, the sensitivity, namely the inhibition rate, is the concentration IC of the analyte corresponding to 50 percent50The fluorescence value can be read from the standard curve, and is measured at 560nm for each reaction cup with a fluorescence detector at 365nm excitation wavelength.
7. The kit for the rapid detection of the carbon quantum dot fluorescence immunoassay of the six veterinary drug antibiotics in the poultry tissues according to claim 6, wherein the complex solution of the sample after the comprehensive pretreatment is obtained by the following steps: (1) weighing 2.0g of sample into a 50mL centrifuge tube, adding 5mL of acidic sample extracting solution I and 150 μ L of 2-nitrobenzaldehyde derivatization reagent, and carrying out vortex oscillation for 5 min; (2) placing in a 75-80 ℃ water bath kettle, incubating for 15min in a dark place, taking out, cooling to room temperature, adding 5mL of alkaline extract II and 8mL of ethyl acetate, vortex and shaking for 5min at 4000rpm, taking 2mL of centrifuged supernatant for 5mL in a 5mL centrifuge tube after 5min, and drying with air at 60 ℃ for 10-15 min; (3) adding 2mL of n-hexane and 1mL of phosphate complex solution, and carrying out vortex for 10s at 4000rpm for 3 min; (4) and taking down the layer to be tested.
8. The kit for rapidly detecting carbon quantum dot fluorescence immunoassay of six veterinary drug antibiotics in poultry tissues according to claim 6The method for detecting the object is characterized in that the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb) X 100% where F0For detecting the fluorescence value of the cuvette in the presence of a solution of the substance to be detected, FxFor detecting the fluorescence value of the cuvette in the detection of a solution containing an analyte, FbThe method is characterized in that the inhibition rate of the standard solution of the object to be detected is taken as the ordinate, the logarithm of the concentration of the standard substance of the object to be detected is taken as the abscissa, the standard curve is made for the detected fluorescence value of the reaction cup when the standard solution of the object to be detected or the sample to be detected is not added, the inhibition rate can be calculated by measuring the fluorescence value of the sample according to the concentration of each sample, and the inhibition rate is read from the standard curve according to the inhibition rate.
9. The method for detecting the analyte by using the carbon quantum dot fluorescence immunoassay rapid detection kit for the six veterinary drug antibiotics in the poultry tissues as claimed in claim 7, wherein the detection sensitivity of the kit to furaltadone is 0.12 ng/mL; the detection sensitivity of the furacilin is 0.18 ng/mL; the detection sensitivity of the nitrofurantoin is 0.35 ng/mL; the detection sensitivity of furazolidone is 0.25 ng/mL; the detection sensitivity of the chloramphenicol is 0.12 ng/mL; the detection sensitivity of the florfenicol is 0.032 ng/mL.
10. The method for detecting the analyte by using the carbon quantum dot fluorescence immunoassay rapid detection kit for six veterinary drug antibiotics in the poultry tissues as claimed in claim 7, wherein the six analytes have no cross interference with each other, the cross reaction rate is less than 1%, and the cross reaction rate is as follows: cross reaction rate (CR%) ═ IC50x/IC50A100% of wherein IC50xThe concentration, IC, of the test substance at 50% inhibition50AThe concentration of any one of the five targets except the test object at which the inhibition rate is 50% corresponds to the concentration of the target.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1885038A (en) * | 2006-07-11 | 2006-12-27 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN101241132A (en) * | 2008-01-18 | 2008-08-13 | 华南农业大学 | Nitrofurans medicament metabolite residue ELISA kit and use method |
| CN107957492A (en) * | 2017-11-18 | 2018-04-24 | 安徽师范大学 | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
-
2021
- 2021-09-16 CN CN202111087022.2A patent/CN113884680A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1885038A (en) * | 2006-07-11 | 2006-12-27 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN101241132A (en) * | 2008-01-18 | 2008-08-13 | 华南农业大学 | Nitrofurans medicament metabolite residue ELISA kit and use method |
| CN107957492A (en) * | 2017-11-18 | 2018-04-24 | 安徽师范大学 | A kind of method of the fluorescence immunoassay determining adsorption ultra trace oleyl amine scion grafting polysuccinimide based on carbon dots mark |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
Non-Patent Citations (2)
| Title |
|---|
| BAOLEI DONG等: "Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs", FOOD CHEMISTRY, vol. 294, pages 347 * |
| 张慧洁等: "分子印迹碳量子点荧光探针的合成及其在氯霉素检测中的应用研究", 分析试验室, vol. 38, no. 5, pages 596 - 600 * |
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